The role of endothelin in mediating virus-induced changes in endothelin

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The role of endothelin in mediating virus-induced changes in endothelin
Copyright #ERS Journals Ltd 1999
European Respiratory Journal
ISSN 0903-1936
Eur Respir J 1999; 14: 92±97
Printed in UK ± all rights reserved
The role of endothelin in mediating virus-induced changes in
endothelinB receptor density in mouse airways
P.J. Henry*, M.J. Carr*, R.G. Goldie*, A.Y. Jeng**
The role of endothelin in mediating virus-induced changes in endothelinB receptor density in mouse airways. P.J. Henry, M.J. Carr, R.G. Goldie, A.Y. Jeng. #ERS Journals Ltd
ABSTRACT: Emerging evidence supports a mediator role for endothelin (ET)-1 in
airway diseases including asthma. Respiratory tract viral infections, are associated
with increased levels of ET and altered ET receptor density and function in murine
airways. To determine whether these virus-induced effects are causally linked,
perhaps involving ET-1-induced ETB receptor downregulation, the current study
investigated the influence of in vivo administration of CGS 26303, an ET-converting
enzyme inhibitor, on virus-induced changes in ET-content and ETB receptor density.
CGS 26303 (5 mg.kg-1.day-1) or placebo was administered to mice via osmotic
minipumps implanted subcutaneously. Two days after implantation, mice were
inoculated with influenza A/PR-8/34 virus or sham-infected, and all measurements
were performed on tissue obtained on the fourth day post-inoculation.
Viral infection was associated with elevated levels of immunoreactive ET and
decreased densities of ETB receptors in murine airways. Both of these effects were
attenuated in virus-infected mice that had received CGS 26303. Virus-induced
increases in wet lung weight were also inhibited by CGS 26303. Importantly,
administration of CGS 26303 had no effect on the titres of infectious virus in the lungs
and similarly, viral infection had no effect on the plasma levels of free CGS 26303.
In summary, CGS 26303 inhibited the virus-induced changes in both immunoreactive endothelin content and endothelinB receptor density. These findings are
consistent with the postulate that the elevated epithelial expression of endothelin-1
during respiratory tract viral infection is a contributing factor in the downregulation
of endothelinB receptors in airway smooth muscle. Whether inhibitors of endothelin
synthesis attenuate virus-induced exacerbations of asthma or airways hyperresponsiveness remains to be established.
Eur Respir J 1999; 14: 92±97.
The levels of endothelin (ET)-1 in the airways are significantly elevated in respiratory diseases such as asthma
[1] as well as in several animal models of airways disease,
including allergic inflammation [2, 3] and respiratory
tract viral infection [4]. ET-1, via its potent actions on a
raft of different cell types within the airways and lungs
(for review see [5]), may contribute significantly to airway wall remodelling, oedema, bronchial obstruction,
long-lasting bronchoconstriction and the development of
airway hyperresponsiveness in asthma and during respiratory tract viral infections.
Respiratory syncitial virus induces the expression of
ET-1 in bronchial epithelial cells [6] and the levels of
immunoreactive (ir)-ET-1 are elevated in murine airways
and lung during influenza A viral infection [4]. Increases
in the production of ET-1 by the airway epithelium would
be expected to lead to enhanced stimulation of ETA and
ETB receptors in adjacent tissues such as the airway
smooth muscle. However, respiratory tract viral infection
in mice was associated with reductions in ETB receptor
density, which was reflected in attenuated ETB receptormediated contractile function [7, 8]. This raises the intriguing possibility that the enhanced levels of ir-ET
*Dept of Pharmacology, University of
Western Australia, Australia. **Novartis
Institute for Biomedical Research, Summit, New Jersey, USA.
Correspondence: P.J. Henry
Dept of Pharmacology
University of Western Australia
Nedlands, 6907
Western Australia
Fax: 61 893463469
Keywords: Endothelin-1
endothelin-converting enzyme inhibitors
endothelin receptors
influenza A virus
tracheal smooth muscle
Received: January 27 1999
Accepted after revision March 29 1999
This study was supported by the National
Health and Medical Research Council
(NH&MRC) of Australia, the Asthma
Foundation of Western Australia, the
Medical Fund of Western Australia and
the Raine Foundation.
contribute to the reduction in ETB receptor density and
function, perhaps associated with ET-induced ETB receptor downregulation. In support of this postulate, ETB receptors present in murine and rat tracheal smooth muscle
were readily desensitized in vitro [9]. To further test the
link between virus-induced increases in ir-ET and reductions in ETB receptor density, mice in the current study
were treated with an ET-converting enzyme (ECE) inhibitor CGS 26303 to attenuate virus-induced increases in
ET. If the postulate holds, then administration of CGS
26303 should be associated with inhibition of virusinduced changes in ETB receptor density as well as ET
Drug administration
Eight-week-old male CBA/CaH mice, specified pathogen free, were obtained from the Animal Resources Centre
(Perth, Australia), housed in a controlled environment
(Microbiology Animal House, University of Western
Australia, Nedlands, Australia) and received food and
water ad libitum. Mice were anaesthetized (60 mg.kg-1
pentobarbitone sodium, (i.p.)), and an osmotic minipump
(Alzet 1007D; Alza Corporation, Palo Alto, CA, USA; 7
day duration) containing CGS 26303 or placebo (0.25 M
NaHCO3) was implanted subcutaneously on the back immediately posterior to the scapulae. CGS 26303 was
dissolved in 0.25 M NaHCO3 at 8.75 mg.mL-1 and was
delivered at a rate of 0.5 mL.h-1 (~5 mg.kg-1.day-1).
Respiratory tract virus stock and animal inoculation
Influenza A/PR-8/34 virus was grown in the allantoic
fluid of 10-day-old embryonated chicken eggs at 378C for
3 days as described previously [10]. The allantoic fluid
was harvested and contained 2.76106 mL-1 egg infectious doses (EID50) of virus as determined by the method
of allantois-on-shell titration for infectivity [10]. The
virus stock was stored in 0.5-mL aliquots at -858C. Two
days after implantation of osmotic minipumps, mice
were anaesthetized with Penthrane (methoxyflurane, 1
mL added to a 500 mL-sealed container; Medical Developments, Melbourne, Australia) and groups of CGS
26303-treated animals and placebo-treated controls were
intranasally inoculated with 15 mL of fluid containing
1,000 EID50 doses of influenza A virus. The remaining
mice were sham-infected using a 15 mL solution of a 1:40
dilution of allantoic fluid from virus-free chicken eggs.
On day six of the study (i.e. day four post-inoculation),
mice were sacrificed by an overdose of pentobarbitone
sodium (200 mg.kg-1, i.p; Rhone Merieux Australia Pty
Ltd., Pinkemba, Australia). A 0.4-mL blood sample was
taken to determine the levels of free CGS 26303 in the
circulation. Plasma samples were centrifuged in Centrifree tubes (Amicon, Beverly, MA, USA) and the concentrations of CGS 26303 in the filtrates were measured
using a neutral endopeptidase 24.11 inhibition assay.
Lungs were blotted dry, weighed and prepared for determination of lung viral titres and extraction of ET. The
trachea from each animal was carefully cleaned of adherent fat and connective tissue and prepared for autoradiography.
Lung virus titres
Lung tissues were homogenized in sterile saline with
glass/glass tissue homogenizers, and the resulting suspension was clarified by centrifugation at 2,0006g for 5 min
at 48C. Infectious virus was assayed by allantosis-onshell titration for infectivity as previously described [10].
Briefly, 666 mm pieces of allantois-on-shell from 11day-old embryonated chicken eggs were incubated in
sterile round bottom tubes containing 0.35 mL of
Standard Medium (SM) and 25 mL aliquots of serial
10-fold dilutions of virus (10-2 to 10-6) in SM. Five
replicates were used at each dilution. Tubes were sealed
and placed in a working horizontal shaker in a 358C room
for 48 h. The fluid from each tube was transferred to a
haemagglutination tray and one drop of 10% washed
goose red blood cells was added to each well. The trays
were shaken and left to stand for 40 min. Positive haemagglutination indicated infection and the EID50 was
calculated by the method of THOMSON [11]. The composition of the SM (pH 7.0) was (in mM): NaCl 137, KCl 8,
CaCl2 7.2, MgCl2 0.52, glucose 1.7, acid-free gelatin 2.0
g.L-1, phenol red 2.5 mg.L-1, penicillin 5 UmL-1, streptomycin 5 mg.mL-1 and amphotericin B 12.5 ng.mL-1.
Autoradiographic studies
Tracheal tubes were submerged in Macrodex (6% dextran 70 in 5% glucose) and frozen by immersion in isopentane, quenched with liquid nitrogen. Serial transverse
sections (10 mm) were cut at -208C and thaw-mounted onto
gelatin/chromealum coated glass microscope slides. These
sections were pre-incubated (2610 min) at 228C in a
buffer (50 mM Tris-HCl, 100 mM NaCl, 0.25% bovine
serum albumin, pH 7.4) containing the protease inhibitor
phenylmethysulphonylfluoride (10 mM), and then in another buffer containing 0.2 nM 125I-ET-1 alone for 2.5 h
(total binding) or in the presence of BQ-123 (selective ETA
receptor ligand; 1 mM) or sarafotoxin S6c (selective ETB
receptor ligand; 100 nM). Nonspecific binding was determined in the combined presence of BQ-123 (1 mM) and
sarafotoxin S6c (100 nM). After 2.5 h, tissue sections were
washed twice for 10 min in buffer, rinsed in distilled water
and dried under a stream of cold dry air. Emulsion-coated
cover slips (Kodak NTB-2; Eastman Kodak Company,
Rochester, NY, USA) were attached to one end of the glass
slides with cyanoacrylate adhesive and incubated for 3
days at 48C. The emulsion-coated coverslips were developed (Kodak Dektol, 1:1; Kodak Australasia Pty Ltd.,
Melbourne, Australia) for 3 min, rinsed for 15 s in dilute
acetic acid (2%) containing hardener (Ilford Hypam; Ilford
Imaging Australia, Melbourne, Australia) and fixer (Ilford
Hypam, 1:4; Ilford Imaging Australia) for 2.75 min. Tissue
sections were then stained for 30 s with Gill's double
strength haematoxylin, dehydrated in ethanol, cleared in
xylene and mounted (DePeX; BDH Laboratory Supplies,
Kilsyth, Australia) for light microscopy.
Autoradiographic grain densities were determined using
a computer-assisted grain detection and counting system
[12]. A total of twelve slides were assessed (46total
binding, 36BQ-123, 36sarafotoxin S6c and 26BQ-123
and sarafotoxin S6c) and each slide contained one tracheal ring from each of 32 mice studied (i.e. eight mice in
each of the four groups). Four estimates of grain density
were made per tracheal ring; three over the tracheal
smooth muscle band and one over a nontissue area in the
airway lumen. Thus, a total of 1,536 fields were analysed (12 slides632 sections64 fields). Autoradiographic
grain densities are expressed as grains.1000 mm-2 and
presented as the mean grain density‹SEM.
Extraction of endothelin from lung tissue
Lungs were homogenized with glass/glass tissue homogenizers in a buffer containing 1M acetic acid and 1
mg.mL-1 pepstatin A at a ratio of 10 mL buffer.g wet
weight-1 of tissue. Lung homogenates were then incubated
in a boiling water bath for 10 min to inactivate proteolytic
enzymes, cooled to 48C and centrifuged at 100,0006g for
20 min. The resulting supernatants were frozen and stored
at -858C. ET was extracted from tissue supernatants as
described previously [13]. Briefly, C18 Sep-Pak cartridges
(Waters, Milford, MA, USA) were pretreated with 5 mL
90% acetonitrile (ACN) in 1% trifluoroacetic acid (TFA)
followed by 5 mL 25% ACN in 1% TFA. Samples (100
Substances used included: 125I-ET-1, ET-1, BQ-123
(cyclo(D-Trp-D-Asp-L-Pro-D-Val-L-Leu)), sarafotoxin S6c
(Auspep, Melbourne, Australia), CGS 26303 ((S)-2-biphenyl-4-yl-1- (1H-tetrazol-5-yl) ethylamino-methyl phosphoric acid) (Novartis Pharmaceuticals Corporation,
Summit, NJ, USA), penicillin, streptomycin, amphotericin
B, pepstatin A, phenylmethylsulphonylfluoride (Sigma
Chemical Co., St Louis, MO, USA).
Statistical analysis
In the current study, four groups of mice were studied in
a typical 262 factorial design. Thus, two-way analyses of
variance (ANOVA) were used to determine virus- and
CGS 26303-induced changes in ir-ET content and ETB
receptor density. The Bonferroni correction was used for
multiple comparisons. A p-value #0.05 was considered
statistically significant. Grouped data are presented as
mean‹SEM or mean (95% confidence interval (CI)).
CGS 26303 plasma levels and infectious viral titres
Following administration by osmotic minipump (5
mg.kg-1.day-1 for 6 days), the plasma levels of CGS
26303 in sham-inoculated mice (257 nM; 95% CI, 190±
357 nM) were not significantly different from levels measured in virus-inoculated mice (162 nM; 95% CI, 105±251
nM; NS). Thus, the plasma levels of CGS 26303 achieved
by osmotic pump administration were not affected by
coincident viral infection. Similarly, the levels of infectious
virus in the lungs of placebo-treated mice (7.46‹0.32 log
EID50 per lung) were not significantly different from levels
measured in CGS 26303-treated mice (6.93‹0.19 log
EID50 per lung; NS). Thus, presence of CGS 26303 had no
significant effect on the levels of infectious influenza A
virus present in the lungs 4 days post-inoculation.
Immunoreactive endothelin content
Ir-ET content in the lungs of virus-infected mice (202‹
39 pg.lung-1) was 210% higher than that measured in
sham-infected mice (66‹18 pg.lung-1, p<0.05) (fig. 1).
However, pretreatment with CGS 26303, significantly
attenuated this virus-induced increase in the levels of
ir-ET (p<0.05, 2-way ANOVA), such that ir-ET levels
ir-ET pg lung-1
Fig. 1. ± Levels of immunoreactive-endothelin (ir-ET) in the lungs of
influenza A virus-infected (Virus) and sham-infected mice (Sham)
during coincident administration of CGS 26303 (h) or placebo (u).
Data are presented as mean‹SEM (n=6±7). *: p<0.05.
were only 75% higher in virus-infected mice (109‹14
pg.lung-1) than in sham-infected mice (62‹13 pg.lung-1)
(fig. 1).
Lung wet weights
On average, lung wet weight was 45‹5% greater in
virus-infected mice (160‹5 mg) than in sham-infected
mice (110‹3 mg; p<0.05) (fig. 2). However, in CGS
26303-treated mice, lung wet weight was increased by
only 17‹5% during viral infection (135‹6 mg in virusinfected lungs versus 116‹2 mg in sham-infected lungs;
fig. 2) (p=0.001, compared with placebo treatment).
EndothelinB receptor densities
The levels of specific 125I-ET-1 binding on tracheal
smooth muscle were similar in all four groups of mice
(two-way ANOVA, table 1), although the relative proportions of ETA and ETB receptors differed between
Lung wet weight g
mL of lung supernatant diluted with 100 mL 1% TFA and
200 mL 50% ACN) were then applied to pretreated C18
Sep-Pak cartridges. After sample application the cartridge
was washed sequentially with 20 mL 25% ACN in 1%
TFA, 10 mL H2O and 10 mL 50% methanol. Ir-ET was
eluted with 16 mL 50% methanol in 4% acetic acid.
Eluted samples were then frozen at -858C and dried under
vacuum in a freeze drier unit (Model FD3; Dynavac,
Melbourne, Australia). Dried samples were reconstituted
in sample buffer supplied with the enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemical Co.,
Ann Arbor, MI, USA) and the assay performed in accordance with the manufacturer's instructions. The antibodies used in this assay cross-react with ET-1, ET-2 and
ET-3, but not with big-ET.
Fig. 2. ± Lung wet weight of influenza A virus-infected (Virus) and
sham-infected mice (Sham) during coincident administration of CGS
26303 (h) or placebo (u). Data are presented as mean‹SEM (n=8). *:
Table 1. ± Influence of CGS 26303 on virus-induced
changes in the densities of endothelin (ET)A and ETB
receptors on murine tracheal smooth muscle
CGS 26303
ET receptor density
Data are shown as mean‹SEM (n=7±8). *: density of autoradiographic grains associated with specific 125I-ET-1 binding.
groups (fig. 3, table 1). Tracheal smooth muscle from
placebo-treated, sham-infected mice contained a greater
proportion of ETB receptors (68‹7% of specific ET
receptors) than ETA receptors (32‹7% of specific ET
receptors) (fig. 3). However, as previously described [7,
8], tracheal smooth muscle from virus-infected mice
contained significantly lower densities of ETB receptors
(fig. 3). Administration of CGS 26303 had no significant
effect on the density of ETB receptors in sham-infected
mice (62‹4% of specific ET receptors), but markedly
attenuated the virus-induced reduction in ETB receptors.
In mice that received CGS 26303, virus infection caused
only a 17‹6% reduction in ETB receptor density compared with the 51‹8% reduction observed in placebotreated mice (p=0.01, fig. 3).
Elevated levels of ir-ET within the airways and lung
have been observed in many disorders of the lung, including respiratory tract viral infection [4]. In the current
study, virus-induced increases in ir-ET levels in the lung
were markedly attenuated by CGS 26303, an ECE in-
ETB receptor density % total
Fig. 3. ± Endothelin (ET)B receptor density as a percentage of total ET
receptor density in tracheal smooth muscle from influenza A virusinfected (Virus) and sham-infected mice (Sham) during coincident
administration of CGS 26303 (h) or placebo (u). ETB receptor
densities were determined in quantitative autoradiographic studies
using 125I-ET-1 and receptor-selective ligands. Data are presented
as mean‹SEM (n=7±8). *: p=0.01.
hibitor that has recently been shown to inhibit ET-1
synthesis in cultured guinea-pig tracheal epithelial cells
[14]. The combination of these data indicate that virusassociated increases in ir-ET content were due, at least in
part, to enhanced synthesis of ET by the airway epithelium, although recent studies indicate that tracheal
smooth muscle cells can also express prepro-ET-1 and
ECE-1 messenger ribonucleic acid (mRNA) [15]. Although the underlying mechanism of virus-induced increases
in ET content is unknown, an increased expression of ET1 mRNA is likely to be involved [6]. Cytokines produced
during respiratory tract viral infection [16], stimulated
prepro-ET-1 mRNA expression and ET-1 release [17] as
well as ECE-1 mRNA expression [18] in human cultured
bronchial epithelial cells. Although virus-induced increases in ECE levels have yet to be demonstrated, examination of the structure of the promoters for the ECE-1a
and ECE-1b gene suggest that it is the latter isoform that
is most likely to be expressed in pathological states [19],
such as respiratory tract viral infection.
Elevated levels of ET-1 are associated with reduced ETB
receptor densities in transplanted [20], congested [21] and
virally-infected [4, 8] lungs. In the current study, CGS
26303 inhibited both the virus-induced increase in production of ir-ET and the reduction in ETB receptor
density. One explanation for these findings is that viral
infection increased the production of ET which then induced ETB receptor downregulation, a direct mechanism
that is consistent with previously published studies showing that the ETB receptor is readily susceptible to desensitization in airway preparations [7, 9, 22]. Whether
this CGS 26303-induced attenuation of virus-induced
loss of ETB receptor density is translated into preserved
ETB receptor-mediated contractile responses in tracheal
smooth muscle must await additional studies.
An alternate, or perhaps additional, mechanism that
should be considered is that the increased production of ET
during viral infection promoted the release of inflammatory cell cytokines and mediators, which in turn caused a
reduction in ETB receptor density. Consistent with this
latter indirect mechanism, ET-1 has demonstrable proinflammatory effects in the airways, including the influx of
eosinophils in a murine model of allergic inflammation
[23]. In addition, various inflammatory cell cytokines, including interleukin (IL)-1 and tumour necrosis factor
(TNF)-a, have been reported to modulate ET receptor
levels [24, 25]. In this regard, it is interesting to note that
CGS 26303 inhibited the virus-induced increase in lung
wet weight, perhaps by blunting the pro-inflammatory effects of ET-1. However, the CGS 26303-induced reduction in virus-induced lung weight may be otherwise
explained by considering haemodynamic influences, such
as a reduction in ET-1-induced venoconstriction and the
associated pulmonary congestion.
The levels of infectious virus measured in the lungs of
mice on day four post-inoculation were not significantly
influenced by concomitant administration of CGS 26303
and, similarly, the plasma levels of CGS 26303 were not
significantly affected by respiratory tract viral infection.
Thus, it is unlikely that the observed changes in the levels
of ir-ET and ETB receptor density in virus-infected mice
treated with CGS 26303 (compared with placebo) were
due to impaired growth of virus in the airways of CGS
26303-treated mice. Similarly, the differential effect of
CGS 26303 on ir-ET and ETB receptor levels in virus- and
sham-infected mice cannot be explained on the basis of
differences in the plasma levels of CGS 26303.
CGS 26303 is a potent inhibitor of neutral endopeptidase (NEP) 24.11 as well as ECE and thus due consideration must be given to the possibility that the effects
observed in the current study may, at least in part, have
resulted from NEP inhibition. NEP is present in the airway
epithelium of several animal species, including humans
[26, 27], and is thought to play a role in the catabolism of
ET-1 [14, 28]. Thus, in the present study, CGS 26303induced inhibition of NEP may have had several effects.
Firstly, NEP may well act as an "endothelinase" and thus
inhibition of NEP by CGS 26303 might be expected to
reduce the breakdown of ET-1 and thus increase its levels.
However, compared to placebo-treated mice, the levels of
ir-ET in CGS 26303-treated mice were either unchanged
(in sham-infected mice) or significantly reduced (virusinfected mice), indicating that inhibition of the production
of ET rather than prevention of its breakdown was the
predominant functional effect of CGS 26303 in the current study. Secondly, inhibition of NEP by CGS 26303
may elevate the levels of other bioactive peptides, with
unknown effects on the ET system. However, although
NEP plays a role in the regulation of lung growth and
maturation in foetal mice [29], the levels of NEP in the
upper airways of adult mice are very low (G. Colasurdo,
personal communication) in comparison to several other
animal species. Indeed, although hitherto untested differences in the activity of NEP in mouse and rat trachea
may explain, at least in part, the recently reported observation that parainfluenza-1 virus significantly attenuates ETB receptor density and function in mouse, but not
in rat trachea [30]. The higher activities of NEP in rat
trachea may lead to greater catabolism of ET-1 and thereby protect the ETB receptor from downregulation. In
summary, although further definitive studies are required,
it appears that within the murine trachea the major
influence of CGS 26303 is on ECE rather than NEP.
The major finding of the current study was that CGS
26303 inhibited the virus-induced increase in immunoreactive endothelin content as well as the decrease in endothelinB receptor density. These findings, together with
other data demonstrating that endothelinB receptors are
readily downregulated, support the argument that the
elevated levels of endothelin-1 present in virally infected
mice caused endothelinB receptor downregulation. Furthermore, the results of the current study using an endothelin-converting enzyme inhibitor will be of strategic
importance in future studies aimed at determining the
mediator role of endothelin-1 in virus-induced airway hyperresponsiveness.
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